DNA Delivery Systems for Gene Therapies

Gene therapies are an emerging class of therapies with huge potential to treat diseases ranging from age-related macular degeneration^1,2 to inherited neurological disorders like Fabry disease.^2,3,4 They work by modifying a patient’s genes to replace a disease-causing gene with a healthy copy; deactivate a disease-causing gene; or introduce a new or modified gene into the body to help treat the disease.⁵

Modifying a patient’s genes requires the safe and efficient delivery of genetic material into their cells. This is commonly done using a carrier, such as a virus, which needs to successfully transport DNA without the genetic material degrading or triggering a dangerous immunological reaction.⁶ The most common carriers remain viral vectors, but non-viral methods are also gaining in popularity.

Nearly all of today’s DNA-based gene therapies use viral vectors to deliver genetic material to patients. This is because viruses “have been honed by evolution to be very efficient at delivering genetic material into cells,” explains James Cody, Ph.D., Associate Director Technical Sales and Evaluations, Business Development at Charles River Laboratories. Viruses can also be tailored to target specific cell types.

Three main types of viruses are used: adeno-associated virus (AAV), lentivirus, and adenovirus.⁷

Adeno-associated viruses (AAV)

The leading viral vector for delivering DNA into cells is adeno-associated virus (AAV).⁸ First discovered in 1965, AAV are small viruses that consist of a protein shell surrounding a single-stranded DNA genome.⁹ The DNA genome can be engineered to carry a therapeutic cargo into the cell nucleus without the virus replicating itself.¹⁰

Most adults have antibodies to the adeno-associated virus 2 (AAV2) strain, but don’t have symptoms in most cases.⁹ For this reason, “they are effective vectors for gene therapy applications as they elicit minimal immune response,” explains Carl Christel, Ph.D., Vice President U.S. Operations for Sirion Biotech Solutions at Revvity.

AAV are also able to target specific cell types, unlike currently—for example—lentiviruses, notes Rodney L. Rietze, Ph.D., Co-Founder and CEO of iVexSol. Novartis’ Zolgensma^® gene therapy, for example, targets the brain and motor neutrons to help treat spinal muscular atrophy (SMA),¹¹ a leading genetic cause of infant mortality.^12,13 This makes them ideal for “classic gene therapy or gene-editing approaches, in which a normal copy of a gene is delivered to compensate for a mutation or deletion of some sort,” Dr. Cody adds.

Lentivirus

However, AAV viral vectors also have disadvantages. As Dr. Rietze reports, AAV carriers have been associated with several deaths, raising safety concerns.¹⁴ Moreover, he adds, they can’t be used in dividing cells. This is because the AAV carrier lacks the equipment for viral replication, meaning the DNA isn’t replicated into the cell genome and the new gene is diluted each time the cell divides.¹⁰

In contrast, lentiviruses integrate into the host genome, Dr. Christel says, allowing them to be used where cells are dividing. This means they’re commonly used in gene therapy for making stable cell lines and engineering stem cells to treat immune deficiencies and hemopathologies¹⁵ (disorders involving abnormal production of the hemoglobin molecule¹⁶), Dr. Cody explains.

However, lentivirus have several limitations, including being limited to one gene (cargo) that integrates randomly into the host genome, explains Daniel Fitzgerald, Ph.D., Co-Founder and CEO of Geneva Biotech. This raises the risk of undesirable side effects, such as cancers, if they were introduced directly into the body, which means—Fitzgerald says—that they’re typically used ex vivo to generate large numbers of cells.

Adenovirus

“Adenoviral vectors were the workhorse of gene therapy strategies for many years,” Dr. Cody reports, adding that they can deliver larger (DNA) cargos than AAV. The AAV genome is around 4.8 kilobases (kb) in length, and studies have found that cargos of more than 5 kb tend to reduce yields of the virus.¹⁰ They also give “high gene expression in the widest range of cell types,” he explains.

The disadvantage of adenoviruses is that they tend to elicit an immune response and the triggered expression drops over time, Dr. Christel explains, which makes them undesirable for most gene therapy applications. However, as Dr. Cody notes, they remain useful as vaccine candidates and in oncology settings.

Non-viral vectors

As AAV and adenoviruses can trigger dangerous immunological responses, scientists have turned to non-viral alternatives.⁶ These have multiple benefits, according to Generation Bio, the developers of a cell-targeted lipid nanoparticle (ctLNP) delivery system for gene therapies.¹⁷ Lipid nanoparticles (LNPs) consist of ionized lipids, which can encapsulate genetic material and deliver it inside cells.¹⁸

They are best known as a key component in mRNA COVID-19 vaccines. And, reports Nicholas Barbet, Ph.D., Head of Operation at Vector BioPharma, their successful development for mRNA vaccine delivery has been a primary reason behind the reignited interest in using them for non-viral DNA delivery.

According to Generation Bio, the benefits of ctLNP include treating multiple times with a gene therapy and treating patients with pre-existing antibody immunity to AAV. The company writes that, following a single dose of AAV, antibodies are induced against the protein shell of the virus. Due to this, AAV can only be dosed once, and typically at the upper end of its therapeutic index to maximize the chance of successful treatment.

Taking things forward

Viral and non-viral techniques for DNA delivery are developing rapidly, with a growth in both approved drugs and those in the clinical pipeline, Dr. Cody says. Many companies are working on overcoming the limitations with existing techniques. For example, Generation Bio claim to have developed a closed-ended DNA construct (ceDNA),¹⁹ which they hope will allow them to deliver larger cargos than AAV (e.g., 8 kb).

Other companies are also working on improving AAV safety and DNA cargo capacity. Vector BioPharma, for example, are in preclinical stage development of a Shielded Retargeted Adenovirus-based virus-like particle²⁰ (SHREAD) with better tissue targeting and reduced toxicities compared to AAV, and with a higher DNA cargo capacity (36 kb).

References

1. (Accessed 2023) Gene Therapy for Age-Related Macular Degeneration, Nuffield Department of Clinical Neurosciences: Medical Science Division.

2. (Accessed 2023) Gene Therapy Market Report, MarketsandMarkets™, July 2022.

3. (Accessed 2023) Fabry disease, National Institute of Neurological Disorders and Stroke.

4. Khan, A. et al (2021) Lentivirus-mediated gene therapy for Fabry disease, Nature Communications, Vol. 12 (1178), pp. 1-9.

5. (Accessed 2023) What is Gene Therapy? U.S. Food and Drug Administration, modified 07/25/2018.

6. Ibraheem, D. (2014) Gene therapy and DNA delivery systems. Int. J. Pharm, Vol. 459 (1-2), pp. 70-83

7. Capra, E. et al (2021) Gene-therapy innovation: Unlocking the promise of viral vectors. McKinsey & Company 

8. Wang, D. (2019) Adeno-associated virus vector as a platform for gene therapy delivery. Nature Reviews Drug Discovery, Vol. 18, 258-378 

9. (Accessed 2023) Introduction to Adeno-associated Virus (AAV), Vector Biolabs 

10. Naso, M. F. et al (2017) Adeno-Associated Virus (AAV) as a Vector for Gene Therapy,  BioDrugs, Vol. 31 (4), pp. 317-334

11. (Accessed 2023) Novartis shares Zolgensma long-term data demonstrating sustained durability up to 7.5 years post-dosing; 100% achievement of all assessed milestones in children treated prior to SMA symptom onset, Novartis 

12. Singh, R. N. (2017) Diverse role of survival motor neuron protein. Biochim Biophys Acta, Vol. 1860 (3), pp. 299-315 

13. (Accessed 2023) Zolgensma—how it works, is given, and safety considerations—FAQs 

14. (Accessed 2023) High-dose AAV gene therapy deaths, Nature Biotechnology, Vol. 38 (910) 2020 

15. Staal, F. J. T et al (2019) Autologous Stem-Cell-Based Gene Therapy for Inherited Disorders: State of the Art and Perspectives, Vol. 7 (443) 

16. (Accessed 2023) Hemoglobinopathies: Current Practices for Screening, Confirmation and Follow-up, Centers for Disease Control and Prevention 

17. (Accessed 2023) Our Science: ctLNP, Generation Bio 

18. Jung, H. A et al (2022) Lipid nanoparticles for delivery of RNA therapeutics: Current status and the role of in vivo imaging. Theranostics, Vol. 12 (17), pp. 7509-7531 

19. (Accessed 2023) Our Science: ceDNA, Generation Bio 

20. (Accessed 2023) Technology, Vector BioPharma